Nucleotide excision repair is a DNA repair mechanism that involves the removal of damaged nucleotides from double-stranded DNA followed by closure of the resulting gap. In E. coli, UVRABC nuclease, encoded by the uvrA, uvrB and uvrC genes, catalyzes the removal of UV-induced pyrimidine dimers (and presumably other carcinogen-induced base adducts which distort the DNA double helix) by hydrolyzing the 8th phosphodiester bond 5' and the 4th or 5th phosphodiester bond 3' to the modified nucleotides and removing the resulting 12-13 nucleotide long single-stranded DNA fragment. This project will further characterize UVRABC nuclease and the nucleotide excision repair mechanism in E. coli. Subunit composition of UVRABC nuclease will be determined by sedimentation in glycerol gradients and by gel filtration chromatography. Temperature sensitive and deletion mutants of the uvrA, uvrB and uvrC proteins will be isolated; purified mutant proteins will be used to define the functional domains of the subunits and the role of each in the two incision reactions. The sequences of the uvr proteins will be obtained by DNA sequence analysis and the secondary structures predicted. DNA fragments containing various base adducts will be prepared. The incision sites relative to these adducts will be determined by incubating terminally labeled fragments with UVRABC nuclease and analyzing the reaction products on DNA sequencing gels. Assays measuring the binding, incision, and excision steps of the reaction will be developed to determine the thermodynamic and kinetic parameters governing the interaction of the substrate with the enzyme and its subunits. The chemical groups of the substrate in contact with the enzyme will be identified by alkylation protection and interference methods using a substrate of defined sequence carrying a single pyrimidine dimer. Nucleotide excision repair will be reconstituted in vitro using UVRABC nuclease, DNA polymerase I and E. coli DNA ligase. This system will be used to investigate the origin of the short and long repair patches observed in vivo and the involvement of recA protein in long patch repair.